探索人脐带间充质干细胞（hUMSCs）来源的外泌体的最佳分离条件并对其进行生物学鉴定。

无血清培养法培养hUMSCs，培养上清中收集外泌体，超高速离心法（ult-exo）和沉淀法分离外泌体（pri-exo），Western Blot检测外泌体标志性蛋白CD63、HSP70、HSP90和阴性对照蛋白TAPA1，透射电镜观察分离所得外泌体生物学形态，高清晰质谱分析外泌体包裹蛋白种类。

无血清培养法能够在不改变细胞生物学形态的条件下获取hUMSCs源外泌体，透射电镜下可见超高速离心可以获得具有典型双层膜结构的外泌体，大小在100 nm左右，而沉淀法分离所得产物不具有典型的外泌体形态表征，大小在30 nm左右；Western Blot结果表明超高速离心法所得外泌体CD63、HSP70、HSP90均呈现阳性，阴性对照蛋白TAPA1呈现阴性，沉淀法只能检测到HSP90；高清晰质谱检测出超高速离心法分离所得外泌体有169种蛋白，沉淀法有102种，共有蛋白79种。

超高速离心法和沉淀法所得外泌体在蛋白标志物、生物学形态和内含蛋白种类均有差别，本实验结果提示分离hUMSCs源外泌体超高速离心法优于沉淀法。

To explore the best method of separating exosomes secreted by human umbilical cord mesenchymal stem cells (huMSCS) .

Exosomes were collected from huMSCS in the serum-free medium culture system by either ultracentrifugation or precipitation methods. Western Blot was used to detect TAPA1, CD63, HSP70, and HSP90 of exosomes. Tansmission electron microscope was used to observe exosome morphology, and proteome of exosomes isolated via the two methods was compared.

Exosomes isolated via ultracentrifigation were around 100 nm in diameter under transmission electron microscopy and were positive for CD63, HSP70, and HSP90, while negative for TAPA1; exosomes isolated via 101 Kit were around 30 nm in diameter and were positive only for HSP90. High-resolution mass spectrometry (HRMS) revealed 169 proteins in exosomes isolated via ultracentrifigation while only 102 proteins were identified in exosomes isolated via 101 Kit. Comparative analysis dates from HRMS indicated that there were 79 proteins shared by exosomes isolated by the two methods.

Exosomes isolated by ultracentrifugation and precipitation are different in biomarkers and proteome profile, and ultracentrifugation method is superior to precipitation method.